Diagnostic method for determining content of sialic acid or sialoprotein

ABSTRACT

A hemagglutinate lectin derived from the slug, Limax flavus, having a high degree of hemagglutinative inhibitory specificity and selectivity for sialic acid or sialoproteins and diagnostic and separatory methods and compositions based upon the inhibitory effect of sialoproteins or sialic acid on the hemagglutinating properties of the slug-derived lectin.

This is a divisional of co-pending application Ser. No. 355,378 filedMar. 8, 1982 now U.S. Pat. No. 4,457,865.

BACKGROUND OF THE INVENTION

It has been well documented in the art that many diverse biologicalfunctions are associated with the presence of sialoglycoproteins andsialoglycolipids at or near the cell surfaces in mammals. It has beenshown, for example, that the metastasis of malignant cells may be adirect function of an excessive release by the malignant cells, or byother cells, of sialoproteins or sialopeptides into the blood stream.The following is a list of representative literature referencesdocumenting the correlation between various biological functions and thepresence of at or near the cell surface or the release from the cell ofsialopeptides or sialoproteins: Gandhi et al, Effect of DiabetesMellitus on Sialic Acid and Glutathione Content of Human Erythrocytes ofDifferent Ages, Indian Journal of Experimental Biology, Vol. 17, pp.585-587 (June, 1979); Wautier et al, A Platelet Defect in a Patient withEosinophilic Leukaemia: Ristocetin-Induced Platelet AggregationAssociated with a Reduced Platelet Sialic Acid Content, Scand. J.Haematol., Vol. 22, pp. 267-276 (1979); Sato et al, Target of XIrradiation and Dislocation of Sialic Acid in Decrease of Cell SurfaceCharge of Erythrocytes, Radiation Research, Vol. 69, pp. 367-374 (1977);Buck et al, Sialoglycoprotein Differences Between Xenotransplantable andNonxenotransplantable Ascites Sublines of the 13762 Rat MammaryAdenocarcinoma, Archives of Biochemistry and Biophysics, Vol. 198, pp.12-21 (1979); Dawson et al, Variations in Sialomucins in the Mucosa ofthe Large Intestine In Malignancy: A Quantative and StatisticalAnalysis, Biochemical Journal, Vol. 10, p. 559 (1978); Hakim,Correlation Between Perchloric-Acid-Soluble Serum Proteins, CellularImmunity and Tumor-Cell Burden, Int. J. Cancer, Vol. 25, pp. 281-288(1981); Hassing et al, Ultraviolet Difference Spectral Studies onConcanavalin A-Carbohydrate Interaction, Eur. J. Biochem. Vol. 16, pp.549-556 (1970); Lipton et al, Glycoproteins and Human Cancer, Cancer,Vol. 43, pp. 1766-1771 (1979); Gorman et al, Determination ofProtein-Ligand Equilibria by Difference Spectroscopy. Hemerythrin-LigandThermodynamic Studies, Biochemistry, Vol. 20, pp. 38-43 (1981); Moss etal, Significance of Protein-Bound Neuraminic Acid Levels in Patientswith Protein-Bound Neuraminic Acid Levels in Patients with Prostatic andBladder Carcinoma, Urology, Vol. 13, p. 182 (1979); Codington et al,Variations in the Sialic Acid Compositions in Glycoproteins of MouseAscites Tumor Cell Surfaces, Biochemistry, Vol. 18, pp. 2145-2149(1979); Sherblom et al, Purification of the Major Sialoglycoproteins of13762 MAT-B1 and MAT-C1 Rat Ascites Mammary Adenocarcinoma Cells byDensity Gradient Centrifugation in Cesium Chloride and GuanidineHydrochloride, The Journal of Biological Chemistry, Vol. 255, pp.783-790 (1980); Jumblatt et al, Altered Surface Glycoproteins inMelanoma Cell Variants With Reduced Metastasizing Capacity Selected forResistance to Wheat Germ Agglutinin, Biochemical and BiophysicalResearch Communications, Vol. 95, pp. 111-117 (1980); Vilarem et al,Differences in Sialic Acid Contents of Low Cancer Cells, High CancerCells and Normal Mouse Lung Counterparts, Biochemical and BiophysicalResearch Communications, Vol. 98, pp. 7-14 (1981); Gaffar et al, FurtherStudies on a Human Lung Tumor-associated Antigen, The Journal ofBiological Chemistry, Vol. 254, pp. 2097-2102 (1979); Blithe et al,Comparison of Glycopeptides from Control and Virus-Transformed BabyHamster Kidney Fibroblasts, Biochemistry, Vol. 19, pp. 3386-3395 (1980);Santer et al, Partial Structure of a Membrane Glycopeptide fromVirus-Transformed Hamster Cells, Biochemistry, Vol. 18, pp. 2533-2540(1979); Yogeeswaran et al, Cell Surface Sialic Acid Expression ofLectin-Resistant Variant Clones of B16 Melanoma With AlteredMetastasizing Potential, Biochemical and Biophysical ResearchCommunications, Vol. 95, pp. 1452-1460 (1980); Glick, MembraneGlycopeptides from Virus-Transformed Hamster Fibroblasts and the NormalCounterpart, Biochemistry, Vol. 18, p. 2525 (1979); Allen et al,Glycoprotein Receptors for Concanavalin A Isolated from Pig LymphocytePlasma Membrane by Affinity Chromatography in Sodium Deoxycholate;Nature New Biology, Vol. 236, pp. 23-25 (1972); Hayman et al,Purification of Virus Glycoproteins by Affinity Chromatography UsingLens Culinaris Phytohaemagglutin, Febs, Letters. Vol. 29, pp. 185-188(1973) Lotan et al, Activities of Lectin and Their ImmobolizedDerivatives in Detergernt Solutions. Implications on the Use of LectinAffinity Chromatography for the Purification of Membrane Glycoproteins,Biochemistry, Vol. 16, pp. 1787-1794 (1977).

Lectins are a group of proteins that often demonstrate high bindingspecificity toward carbohydrate residues of glycoproteins andglycolipids. They have proven invaluable in the study of glycoproteinsof cell surfaces as well as the modifications they undergo during celldifferentiation and malignant transformation. Relatively few lectinswith specificity for sialic acid have been identified. Two such lectins,limulin and carcinoscorpin, have been purified from the Americanhorseshoe crab, Limulus polyphemus, and the Indian horseshoe crab,Carcinoscorpius rotunda cauda, respectively. Limulin also bindsN-acetylglucosamine and D-glucuronic acid. The carbohydrate specificityof carcinoscorpin has not been extensively studied. A lectin, LAg-1,which binds N-acetylneuraminic acid, N-glycoylneuraminic acid andN-acetylmannosamine has been isolated from the lobster, Homarusamericanus. A lectin which binds sialic acid residues of glycoproteinshas also been isolated from wheat germ. Agglutination of erythrocytes bywheat germ agglutinin is inhibited by N-acetyl-D-galactosamine andN-acetylneuraminic acid, with N-acetyl-D-glucosamine being the morepotent inhibitor.

It has been reported that extracts of the slug, Limax flavus,agglutinate red blood cells (Pemberton, Vox Sang., Vol. 18, ppl 74-76(1970); however, no carbohydrate specificity was defined for theagglutinin.

The specific sialoprotein binding lectin (limulin and carcinoscorpin) ofthe prior art are relatively unstable, however, due to their largemolecular size and multisubunit composition (18 to 20). Moreover, due tothe scarcity of the sources for the lectins, the latter are expensive toprepare or procure.

It is an object of the present invention to provide a highly specificsialic acid binding lectin.

It is a further object of the present invention to provide methods forobtaining homogeneous preparations of this lectin from the slugs, Limaxflavus.

It is a further object of the present invention to provide methods forquantitating the amount of sialoproteins or sialopeptides present inbiological fluids or cellular membranes.

It is a further object of the present invention to provide and/ordescribe methods for the separation and comparison of the sialoproteinsand sialopeptides present in biological fluids or solubilized membraneproteins.

It is a further object of the present invention to provide methods forthe purification of sialoproteins and sialopeptides in quantitiessufficient for antibody preparation to said sialoproteins orsialopeptides.

SUMMARY OF THE INVENTION

The present invention is predicated on the discovery that a lectinisolated from the slug, Limax flavus, has a high degree of specificityand selectivity for binding sialic acid of sialoproteins andsialopeptides. The lectin has a high degree of hemagglutinative activitywhich is selectively inhibited by sialic acid or sialoproteins.

This hemagglutinating activity of the slug-derived lectin which isselectively inhibited by sialic acid or sialoproteins as opposed toother carbohydrates normally present in mammal biological liquidsenables the provision of compositions and diagnostic methods fordetermining the amount of sialopeptides or sialoproteins present in abiological liquid.

The affinity of the lectin for the sialic acids or sialoproteins alsoenables the provision of a method for separating sialopeptides residuesor sialoproteins from their admixtures with other carbohydrates andproteins.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions apply with respect to the terms used hereinand in the appended claims:

"Slug"--the slug, Limax flavus.

"Sialic acid"--the generic name for the family of the N- andO-substituted derivatives of neuraminic acid, e.g., N-acetylneuraminicacid, N-glycolyneuraminic acid and other analogous N- or O-acylatedderivatives of neuraminic acid.

"Sialoproteins"--any of the glycoproteins found in mammals containingsialic acid residues.

"Sialopeptides"--any sialic acid containing peptide derived fromsialoproteins by cleavage with proteases or by other means.

"Lectin" or "agglutinin"--refers to a group of proteins which bind moreor less specifically to carbohydrate residues or glycoproteins.

"LFA"--refers to the sialic acid specific lectin or agglutinin of theslug, Limax flavus.

"Hemagglutinating inhibitory specificity and selectivity for sialic acidor sialoproteins"--refers to the inhibitory effect of sialic acid andsialoproteins on the hemagglutinating properties of the slug-derivedlectin as opposed to the relative lack of inhibitory effect of othercarbohydrates normally found in mammals on the hemagglutinating activitythereof.

"Ammonium sulfate fractionation"--refers to the conventional method forfractionating proteins from biological liquids based upon the relativeinsolubility of various protein fractions in ammonium sulfate solutionsof varying concentration. (See A. A. Green and W. L. Hughes, Methods inEnzymology (1955) Vol. 1, pp. 32-56, ed. by S. P. Colowick and N. O.Kaplan.)

"Biological liquid or fluid"--refers to any liquid or suspension derivedfrom a mammal, e.g., blood, plasma, serum, urine, tissue homogenate,etc.

"Lectin-sialic acid" or "lectin-sialoprotein complex"--refers to thecomplex or conjugate formed by the specific binding of the sialic acidor sialic acid residue of the sialoprotein to the slug-derived lectin.

The sialic acid or sialoprotein specific lectin of the present inventionis derived from the slug, Limax flavus, by homogenization of the slugtissue to release the lectin from the slug tissues with which it isassociated and is partially purified by ammonium sulfate fractionation.

Following the ammonium sulfate fractionation, the lectin fraction may befurther purified to homogeneity by affinity chromatography on a sialicacid containing protein bound to an inert substrate such as sepharose,etc. to yield a homogeneous slug lectin preparation having theapproximate amino acid composition:

    ______________________________________                                                           Moles per 22,000 g of lectin                               Amino Acid         (Nearest Integer)                                          ______________________________________                                        Asx (asparagine + aspartic acid)                                                                 26                                                         Thr                13                                                         Ser                13                                                         Glx (glutamine & glutamic acid)                                                                  14                                                         Pro                4                                                          Gly                22                                                         Ala                16                                                         Val                7                                                          Met                2                                                          Ile                7                                                          Leu                15                                                         Tyr                10                                                         Phe                10                                                         His                7                                                          Lys                17                                                         Trp                6                                                          Arg                8                                                          Cys                6                                                          ______________________________________                                    

It will be understood by those skilled in the art that the approximateamino acid composition set forth herein above is accurate to withinabout ±10% with respect to the various amino acid contents set forth.

The high specificity and selectivity of the slug derived lectin forsialic acid and sialoproteins enables its use as an agent for separatingor isolating sialoproteins or sialopeptides from other proteins orpeptides and glycoproteins in biological liquids or other solutions orsuspensions. The present invention provides a method for isolatingsialopeptides or sialoproteins from a biological or non-biologicalliquid comprising contacting the liquid with the above described lectinto form a lectin-sialopeptide or lectin-sialoprotein complex from theliquid by, e.g., centrifugation to collect the lectin-sialoprotein orlectin-sialopeptide precipitate. The amount of protein in theprecipitate can be quantitated by analyzing the precipitate for proteincontent by any of several commonly used procedures such as the Lowry,Nesseler or the Coomassie blue procedure of BioRad. The amount ofsialoproteins and/or sialopeptides present in biological fluids can alsobe determined by determining the ability of the fluids to inhibit theinteraction of a labeled sialoprotein or sialopeptide with the lectin inradioimmunoassays or Elisa type assays. Alternatively, the sialoproteinsor sialopeptides present in the precipitate can be solubilized in asodium dodecyl sulfate and β-mercaptoethanol containing buffer andseparated by the sodium dodecyl sulfate-polyacrylamide gel method ofLaemmli (Laemmli, U.K., Nature (London) Vol. 227, pp. 680-685, 1970).The sialoproteins or sialopeptides are separated according to size andcan be visualized on the gel by staining the protein bands with proteinor carbohydrate specific stains such as Coomassie blue or periodic acidSchiff's base staining procedures. In this way the sialoproteins orsialopeptide composition of various biological sources can be compareddirectly.

A preferred method of separation of sialoproteins or sialopeptides, inquantities sufficient for characterization, from biological liquidsinvolves affinity chromatographic techniques whereby the slug-derivedlectin is bound to an insoluble matrix, such as sepharose, Sephadex orpolyacrylamide, inert to the biological liquid and suitable for affinitychromatography and contacting the biological liquid therewith in anaffinity chromatographic column. The lectin can be coupled to aninsoluble matrix by any of a number of published procedures. Oneconvenient method is to couple the slug lectin to Sepharose 4B accordingto the procedure of Marsh et al (March, S. C., Parikh, 11, andCuatrecasas, P., Anal. Biochem., Vol. 60, pp. 149-152, 1973), wherebycyanogen bromide is used to activate Sepharose 4B with subsequentinteraction of the lectin with the activated sepharose.

The lectin-Sepharose 4B complex is poured into a chromatographic columnand washed free of any noncoupled lectin. A liquid containingsialoproteins or sialopeptides is run through the column. Sialoproteinsand sialopeptides are bound by the lectin and retained in the columnwhereas other proteins or peptides wash through the column. Thesialoproteins and sialopeptides can be recovered by washing the columnwith a liquid containing sialic acid and thus separated from the othercomponents present in the original liquid. Furthermore, thesialoproteins can be fractionated by eluting them from the column with abuffer containing increasing concentrations of sialic acid, i.e., bygradient elution. The recovered sialoproteins can be furtherfractionated by any of several commonly used protein fractionatingprocedures such as: ion exchange chromatography, sieve chromatography,isoelectric focusing or chromofocusing or by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This will permit a comparison of thesialopeptides or sialoproteins of biological fluid derived from thedifferent individuals or sources.

The high degree of specificity of the slug lectin for sialic acid andsialoproteins enables the provision of a diagnostic method forquantitating the amount of sialic acid or sialoproteins in a biologicalliquid by determining its inhibitory effect on the agglutination oferythrocytes by the slug lectin. Thus, the method according to thepresent invention comprises contacting the said biological liquid with apredetermined quantity of the lectin described above and measuring thedegree of hemagglutination effected by said lectin and comparing saidmeasurement with the degree of hemagglutination effected by contactingan identical quantity of said lectin with a similar biological fluidcontaining a known amount of sialic acid or sialoprotein.

The degree of hemagglutination is preferably measured by determining theoptical density at 620 nm of the lectin-treated biologicalliquid-erythrocyte suspension and comparing the optical density thereofwith control samples containing a known sialopeptide or sialoproteinstandard.

The present invention also provides a composition suitable for thediagnostic determination of the sialic acid content of the biologicalliquid of a mammal comprising an aqueous solution or suspension of anamount of the lectin described above sufficient to exert a measurablehemagglutinate effect on the biological liquid sample.

The aqueous solution or suspension preferably comprises isotonic salineand contains an amount of the slug-derived lectin sufficient to exert ameasurable hemagglutinate effect.

The hemagglutinating activity of the slug lectin is highly specific forsialic acid. Of some 17 carbohydrates tested for their ability toinhibit hemagglutination by purified slug lectin, onlyN-acetylneuraminic acid and N-glycolylneuraminic acid were found to beeffective. N-acetylneuraminic acid and N-glycolylneuraminic acid gave a50% inhibition of hemagglutination at 0.13mM and 0.90mM, respectively.Further evidence for the sialic acid specificity of the slug lectin hasbeen obtained by studies with the sialoproteins, fetuin and bovinesubmaxillary mucin (BSM). BSM at ˜9 μg/ml and fetuin at ˜50 μg/ml gave a50% inhibition of the lectin hemagglutinating activity. Treatment of thesialoproteins with neuraminidase or 0.1N H₂ SO₄ resulted in a loss ofinhibitory activity which was proportional to the loss of sialic acidfrom these sialoproteins. Thus, the inhibition of slug lectinhemagglutinating activity by these sialoproteins appears to be duesolely to their sialic acid residues.

The slug lectin is a relatively small molecule, consisting of twosubunits of ˜22,000 Daltons each, and is highly specific for sialic acidresidues of sialoproteins. The lectin is particularly useful in thestudy of membrane structure and differences between normal and malignantcells as well as in the purification of sialoproteins and the comparisonof the sialoprotein content of different biological fluids, such asserum, plasma, urine or tissue homogentates from normal and cancerpatients.

DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a plot showing the agglutination assay for the slug lectin.

FIG. 2 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis ofthe lectin.

FIG. 3 is a plot of hemagglutinin activity against various fractions ofprotein.

FIG. 4 is a plot of sedimentation velocity behavior of the lectin.

FIG. 5A is the circular dichroic and absorption spectra (near UV) of thelectin.

FIG. 5B is the circular dichroic and absorption spectra (for UV) of thelectin.

FIG. 6 is the sodium dodecyl sulfate polyacrylamide gel electrophoresisof lectin-sepharose affinity fractions from serum.

EXAMPLES

Unless otherwise stated, all purification procedures were carried out atapproximately 5° C.

Hemagglutination Assay: Aliquots of lectin were adjusted to 0.5 ml byaddition of tris-saline buffer which contained 0.4 mg/ml gelatin. Tothis was added 0.5 ml of a solution containing human erythrocytes thathad been washed several times with 0.9% NaCl and whose absorbance at 620nm was ˜2. After standing at room temperature for 30 min. the cells weregently pelleted by centrifugation in a Dynac centrifuge. The cells wereresuspended by shaking and allowed to stand for 5 min. in order topermit aggregated cells to settle. The absorbance at 620 nm of the upperone-half ml of the erythrocyte suspension was measured and the data wereplotted as A₆₂₀ versus μg or μl of lectin. One unit of activity isdefined as the amount of LFA which gave a 50% agglutination. Percentagglutination was calculated as follows: ##EQU1##

Coupling of Bovine Submaxillary Mucin (BSM)¹ to 4B-Sepharose: Twenty mlof 4B-Sepharose were washed with 200 ml of deionized water byfiltration, and the excess water was removed. The Sapharose wassubsequently washed with 20 ml of 2M potassium phosphate (pH 12) byfiltration and then placed in another ml of the phosphate buffer. Twoand one-half ml of dioxane containing 1.0 g of cyanogen bromide wereadded over a 6 min. period while stirring the Sepharose in an ice bath.The Sepharose was stirred for an additional 10 min. and washed with 400ml of deionized water. Ten ml of BSM (10 mg/ml) in 1.0M NaCl and 10 mlof 0.2M NaHCO₃, pH 9.0, were added to the activated Sepharose and themixture stirred for 2 hr. at 22°. The BSM-Sepharose was washed with 100ml of 1.0M NaCl and 100 ml of tris-saline buffer (0.05M tris-Cl-0.10MNaCl, pH 7.5 at 5° C.). The absorbance of the washes at 280 and 260 nmindicated that approximately 85% of the BSM had been coupled to theSepharose.

Purification of Slug Lectin: Homogenates of whole slugs were highlyviscous and were only partially clarified by centrifugation at 30,000×gfor 30 min. Ammonium sulfate precipitates from such homogenates yieldedhighly viscous solutions when resuspended in buffer; the high viscosityof these solutions greatly hampered further purification of the LFA. Theproblems associated with highly viscous solutions can be eliminated byevisceration of slugs and use of only the body tissues for the lectinpreparation. About 90% of the total agglutination activity wasassociated with the body tissues, about 5-7% was associated with thehemolymph and less than 2% was associated with the internal organs.Furthermore, most of the viscous material (presumably mucins) wasassociated with the viscera and thus was eliminated by this procedure.

Approximately 150 g of slugs were washed with deionized water,eviscerated, and further washed with tris-saline buffer to removesurface mucous material. One-hundred g of washed tissue were placed in400 ml of tris-saline buffer containing 4.0 ml ofphenylmethylsulfonyl-fluoride (5 mg/ml in 2-propanol). The tissue wasminced with scissors and then homogenized by use of a polytronhomogenizer at full speed for 3 min. The homogenate was centrifuged at16,000×g for 15 min. And the supernatant fraction saved (SF-16).Powdered ammonium sulfate was added to the SF-16 to 40% saturation andthe precipitate collected by centrifugation at 16,000×g for 15 min. Thesupernatant fraction was decanted and adjusted to 80% saturation byadditions of powdered ammonium sulfate. The precipitate (AS-40-80) wascollected by centrifugation at 16,000×g for 15 min. . The AS-40-80pellets were resuspended in 200 ml of tris-saline buffer and dialyzedagainst three-one liter volumes of this buffer at 5° for 24 hrs.

A 1.6×10 cm column of BSM-Sepharose was equilibrated with tris-salinebuffer. One-hundred ml of the dialyzed AS-40-80 fraction atapproximately 10 mg of protein per ml were pumped (50 ml/hr) onto thecolumn. The column was washed with tris-saline buffer until thenon-bound proteins were eluted. The lectin was then eluted withtris-saline buffer containing 10mM AcNeu. The fractions containingprotein which eluted with AcNeu were combined and dialyzed againsttris-saline buffer before storage at -70°.

As indicated in Table 1, most of the agglutinin activity present in thetissue homogenate was recovered in the 40-80% ammonium sulfate fraction.Subsequent purification by affinity chromatography of LFA onBSM-Sepharose (FIG. 1) resulted in a 68-fold overall purification of LFAwith a 22% recovery of agglutinin activity (Table 1). The use of BSM inconjunction with AcNeu as an agent in the affinity purification of LFAresults in the recovery of a lectin which binds AcNeu. The low recovery(22%) and the apparently rather low degree of purification (68-fold) ofLFA is probably due to a separation, at the affinity chromatographystep, from other hemagglutinins which are specific for carbohydrateresidues other than sialic acid, a suspicion confirmed by the datadiscussed below. In spite of the low recovery of hemagglutinin activity,approximately 17 mg of purified LFA was obtained from 100 g ofeviscerated slug tissue.

                  TABLE I                                                         ______________________________________                                        Purification of a Sialic Acid Specific Slug Lectin.sup.a                                             Specific                                                      Vol-            Activity.sup.c                                                                         Total    Re-                                         ume     Protein (Units/mg                                                                              Activity covery                               Fraction.sup.b                                                                       (ml)    (mg)    protein) Units × 10.sup.-3                                                                (%)                                  ______________________________________                                        SF-16  434     5034     62      309      100                                  AS-40-80                                                                             104      946    169      334      108                                  Purified                                                                              28      16.8   4169      70       22                                  LFA                                                                           ______________________________________                                         .sup.a Onehundred g of eviserated slug tissue were used in the                .sup.b The following notations are used: SF16, the supernatant fraction       obtained from a 16,000 × g centrifugation of the tissue homogenate;     AS40-80, proteins which precipitated between 40 and 80% saturation of SF1     with ammonium sulfate; purified LFA, protein eluted from the BSMSepharose     column with AcNeu, i.e., affinity purified                                    .sup.c One unit of activity is defined as that amount of lectin which         gives a 50% agglutination of erythrocytes in the hemagglutination assay. 

Removal of Bound AcNeu From Purified Lectin: Extensive dialysis ofaffinity purified lectin against tris-saline did not completely removeAcNeu. Assays of dialyzed lectin by the thiobarbituric acid assay ofWarren, J. Biol. Chem., 234, 1971-1975 (1959) indicated that the lectincontained approximately eight moles of non-covalently bound AcNeu permole of lectin. Chromatography of the lectin on a column (1×37 cm) ofDowex 501-X8(D) which had been equilibrated with 0.01M ammonium acetate,pH 7.0, reduced its AcNeu content to less than 0.20 moles of AcNeu permole of lectin with little or no change in the specific activity of thelectin preparation. Because the yield of protein from such a treatmentwas ˜70% and because the removal of AcNeu had no effect on the lectin'sspecific activity, samples of lectin which had been chromatographed onthe ion exchange column were utilized for only selected experiments.

Inhibition of Hemagglutination: An amount of purified LFA or partiallypurified LFA (i.e., AS-40-80 fraction) which gave greater than 95%agglutination of erythrocytes in the hemagglutination assay was mixedwith varying amounts of potential inhibitors dissolved in tris-salinebuffer, and the volume was adjusted to 0.5 ml by addition of tris-salinebuffer. A 0.5 ml aliquot of erythrocytes was added to the lectin plusinhibitor solution and the percent agglutination determined as describedabove. The percent inhibition of agglutination represents the differencebetween the percent agglutination with lectin alone and that obtainedwith lectin plus inhibitor.

Chemical Analyses: Amino acid analyses of LFA were determined on acomputerized Durrum 600 amino acid analyzer. Proteins samples werehydrolyzed at 110° in constant boiling HCl for 24, 48 and 72 hrs.Correction were made for loss of threonine and serine during hydrolysis.Cysteine was determined after performic acid oxidation of the protein.Tryptophan and tyrosine were estimated by the spectroscopic method ofEdelhoch, Biochem., 6, 1948-1954 (1967) as modified by Bredderman, Anal.Biochem., 61, pp. 298-301 (1974). AcNeu was measured after acidhydrolysis of neuraminidase treatment of glycoproteins by thethiobarbituric acid assay described by Warren, supra. The acidhydrolytic release of AcNeu from glycoproteins was accomplished byincubation of the glycoprotein in 0.1N H₂ SO₄ at 80° for 1 hr.

Physical Measurements: Sedimentation velocity and equilibriummeasurements were performed in a Beckman model E analyticalultracentrifuge equipped with Schlieren/interference optics and aphotoelectric scanner.

Sedimentation velocity measurements were made according to standardprocedures; no corrections were made for the Johnson-Ogston effect.Meniscus depletion sedimentation equilibrium measurements employed thehigh speed or the long column methods. For all sedimentation equilibriumruns, attainment of equilibrium was checked by measuring the fringedisplacements at several radial distances of two successive exposurestaken 3 to 6 h apart. The partial specific volume used for LFA, ascalculated from its amino acid composition, was 0.724 ml/mg. For LFA atpH 2, a correction was included for the Donnan effect on the equilibriumsedimentation pattern in a three-component system.

Empirical estimations of the hydrodynamic size of native LFA wereattempted by gel chromatography on a 50 cm column of Bio-Gel P-200.Standard proteins of known hydrodynamic properties were utilized tocalibrate elution positions from the column as a function of theequivalent hydrodynamic radius, or of the molecular weight. Empiricalestimations of the size of the constituent polypeptide chains of LFAwere by thin-slab sodium dodecyl sulfate (NaDodSO₄) polyacrylamide gelelectrophoresis and by gel chromatography in the presence of 6M Gdm-Clon Sephacryl S-300.

Circular dichroic spectra were measured on a Cary 60 spectropolarimeterequipped with a model 6002 CD attachment. The CD was calibrated with10-d-camphorsulfonic acid. Cell path lengths from 0.5 to 10 mm wereemployed to maintain optimal signal to noise ratios. A value of 105 wasused for the mean residue molecular weight. The differentialrefractometric method of Babul and Stellwagen, Anal. Biochem. 28, pp.216-221 (1969) was employed to estimate protein concentration.

Absorption spectra were obtained on a Cary 15 spectrophotometer whoseabsorbance accuracies were checked with dichromate.

Homogeneity of Purified LFA: Electrophoresis of the affinity purifiedLFA on NaDodSO₄ -polyacrylamide gels (12.0%), after reduction anddenaturation of the protein by NaDodSO₄ and β-mercaptoethanol, yielded asingle Coomassie Blue staining band (FIG. 2). Electrophoresis wascarried out on 1.5 mm slab gels by the reducing and denaturing method ofLaemmli (1970). Lanes 1 and 5,5 μg each of myoglobin (17,200),chymotrypsinogen A (25,000), and ovalbumin (43,000); lane 2, 10 μg LFA;lane 3, 5 μg LFA; lane 4; 2.5 μg LFA. The migration of LFA relative tothe migration of myoglobin, chymotrypsinogen A and ovalbumin suggeststhat reduced and denatured LFA consists of a single polypeptide speciesof molecular weight approximately 22,000. Some preparations of purifiedLFA exhibited a faster migrating minor component when a relatively largeamount of LFA (>10 μg) was applied to the gel. Velocity sedimentationstudies described herein below provide further evidence of homogeneityof the affinity purified LFA.

Binding Properties of LFA: As indicated in Table II, hemagglutination bythe affinity purified LFA was inhibited by 50% at 0.13mM and 0.81mMconcentrations of AcNeu and GlyNeu, respectively. No inhibition wasobserved with the other carbohydrates tested, even at concentrations ashigh as 10-25mM. In contrast, inhibition of hemagglutination by the40-80% ammonium sulfate fraction by GalNAc, GlcNAc, GalN and GlcN inaddition to AcNeu and GlyNeu (Table II) suggests that this fractioncontains hemagglutinins with binding specificities for carbohydrateresidues other than sialic acid. Those fractions containing proteinswhich did not bind to the affinity matrix were without significanthemagglutinin activity (FIG. 3). Loss of agglutinin activity during thechromatography step may have been due to inactivation of lectins bydenaturation or to loss of essential cations.

                  TABLE II                                                        ______________________________________                                        Inhibition of Hemagglutinin Activity                                          of Slug Lectin by Carbohydrates.sup.a.                                                     Carbohydrate                                                                            Inhibition (%)                                                        Concentration                                                                             AS-40-80 Purified                                  Carbohydrate   (mM)        Fraction LFA                                       ______________________________________                                        N--Acetylneuraminic Acid                                                                     .05         6        1                                                        .10         17       28                                                       .15         27       54                                                       .20         94       100                                       N--Glycolyneuraminic                                                                         .25         3        0                                         Acid           .50         20       2                                                        .75         56       14                                                       1.0         88       85                                        N--Acetyl-D-Galactosamine                                                                    10.0        75       0                                         N--Acetyl-D-Glucosamine                                                                      10.0        53       0                                         Galactosamine  25.0        45       0                                         Glucosamine    25.0        19       0                                         D(+)Galactose  25.0        0        0                                         D(-)Glucose    25.0        0        0                                         D(+)Mannose    25.0        0        0                                         α(+)Arabinose                                                                          25.0        0        0                                         D(+)Xylose     25.0        0        0                                         α-L(-)Fucose                                                                           25.0        0        0                                         D(-)Glucuronic Acid                                                                          25.0        0        0                                         α-Methyl-D-Glucoside                                                                   25.0        0        0                                         α-Methyl-D-Mannose                                                                     25.0        0        0                                         Lactose        25.0        0        0                                         Sucrose        25.0        0        0                                         ______________________________________                                         .sup.a The standard hemagglutination assay was used in these comparative      studies. Twenty μg of protein from the 40-80% ammonium sulfate fractio     (AS40-80) or 0.34 μg of the purified LFA was utilized in each assay.  

As mentioned above, the data of Table II shows that the purified LFAbinds free AcNeu in a highly specific manner. This specificity wasfurther tested by use of the AcNeu-rich glycoprotein, BSM. As shown inTable III, as little as 9 μg/ml of BSM gave a 50% inhibition ofhemagglutination in the standard assay containing 0.34 μg of LFA. Upontreatment of BSM with vibrio cholerae neuraminidase, there was a timedependent release of AcNeu from the BSM with a concomitant loss inagglutination inhibition. The total content of AcNeu in BSM was 5% (w/w)as measured after its release by acid hydrolysis. Release of 12 μg ofAcNeu per mg of BSM, i.e., 25% of its total AcNeu, virtually abolishedits inhibitory activity. Thus, it appears that only about 25% of theAcNeu residues in BSM are the functional ligands in its interaction withLFA. This suggests that BSM may contain AcNeu residues that areinaccessible to LFA.

                  TABLE III                                                       ______________________________________                                        Inhibition of Hemagglutination by                                             Neuraminidase Treated Bovine Submaxillary Mucin.                                           μg BSM Required for 50% Inhibition                            Length of                                                                             AcNeu      BSM Incubation                                                                             BSM Incubation                                Incubation.sup.a                                                                      Released.sup.b                                                                           With         Without                                       (min.)  μg/μg BSM                                                                          Neuraminidase                                                                              Neuraminidase.sup.c                           ______________________________________                                         0      0           9           8                                             15      0.8        22           9                                             30      2.5        33           10                                            45      5.8        40           8                                             60      7.4        70           10                                            120     11.9       >100         9                                             ______________________________________                                         .sup.a A 1.0 mM solution of CaCl.sub.2 containing 2 mg/ml BSM and 38 U of     Vibrio cholerae neuraminidase was adjusted to pH 6.2 and incubated at         37°. Aliquots were removed at times indicated above, diluted 10fol     with .05 M trisCl-.1M NaCl, pH 7.5, and assayed with 0.34 μg slug          lectin for inhibitory activity.                                               .sup.b Aliquots were assayed for released AcNeu by the thiobarbituric aci     assay of Warren, supra.                                                       .sup.c The BSM was incubated as described above except the neuraminidase      was omitted from the incubation mixture.                                 

Physico-Chemical Properties of LFA: The amino acid composition of LFA issummarized in Table IV. The lectin contains 40 residues of Glx plus Asxand a total of 32 residues of Lys, Arg and His per subunit. The highcontent of basic amino acids and the basic pI of the protein suggestthat a relatively large proportion of the Glx and Asx are present in theprotein in the amidated form.

                  TABLE IV                                                        ______________________________________                                        Amino Acid Composition of Slug Lectin.                                                     Moles per 22,000 g.sup.a                                         Amino Acid   Nearest Integer                                                  ______________________________________                                        Asx          26                                                               Thr          .sup. 13.sup.b                                                   Ser          .sup. 13.sup.b                                                   Glx          14                                                               Pro           4                                                               Gly          22                                                               Ala          16                                                               Val           7                                                               Met           2                                                               Ile           7                                                               Leu          15                                                               Tyr             10 (9.6).sup.c                                                Phe          10                                                               His           7                                                               Lys          17                                                               Trp          .sup. (6).sup.c                                                  Arg           8                                                               Cys          .sup.  6.sup.d                                                   ______________________________________                                         .sup.a These values represent the average of values obtained from a 24,       48, and 72 hrs. hydrolysis in constant boiling HCl at                         .sup.b Thr and Ser were corrected for losses during                           .sup.c The values in parentheses for Tyr and Trp were obtained by the         spectroscopic method of Edelhoch, supra, as modified by Bredderman,           .sup.d Cys (halfcystine) was determined by amino acid analysis after          performic acid oxidation of the slug lectin.                             

Although LFA exhibited a single electrophoretic component underdissociating and denaturing conditions (FIG. 2), appreciable sizeheterogeneity was evident during sedimentation velocity measurements atprotein concentrations greater than 1 mg/ml. As illustrated by FIG. 4,these solutions exhibited two boundaries in the analyticalultracentrifuge. The magnitude of the sedimentation coefficient of thefaster sedimenting species exhibited a marked positive dependence on theprotein concentration to the point that at concentrations below 1 mg/mlonly a single species of 3.4 S was observed. The 3.4 S species waspresent under all conditions and its sedimentation coefficient exhibiteda slight positive concentration dependence. The relative ratios of theultracentrifuge species could not be perturbed with AcNeu or chelatingagents (FIG. 4). That these sedimentation velocity data are consistentwith a rapidly associating-dissociating protein system is supported bythe behavior of the near UV circular dichoism spectra of LFA at proteinconcentrations at or above which only the 3.4 S species could beobserved in the analytical ultracentrifuge.

The amplitude of the ellipticity band at 282 nm increased withincreasing protein concentration above ˜0.6 mg/ml; this increaseproduced a marked qualitative change in the near UV spectrum (cf. curves1 and 2 of FIG. 5A). Below ˜0.6 mg/ml, the near UV spectra werequalitatively identical, but the amplitudes of the ellipticity bandsfluctuated slightly among different preparations. In the absorbancerange which could be measured at 280 nm (i.e. protein concentrations ≲1mg/ml), LFA obeyed Beer's Law (absorption spectrum given in FIG. 5A).Thus, the near UV spectral data shows that the increased amplitudes ofthe near UV circular dichroism bands reflect an induced or enhancedchiral environment of one or more aromatic side chains upon associationof lectin molecules. Slight increases in the ellipticity values wereobserved at all wavelengths in the far UV at protein concentrationsabove ˜6.0 mg/ml (Curves 1 and 2 of FIG. 5B), but the magnitudes ofthese increases, up to ˜13%, can be accounted for by the inducedchirality of the aforementioned aromatic amino acid residues.

A cross-linked polyacrylamide gel chromatographic support medium wasemployed in an attempt to utilize it as a purification step and toestimate the equivalent hydrodynamic radius of native LFA. This type ofsupport was employed rather than a gel of polysaccharide matrix todiminish the possibility of lectin-gel interactions. However, theelution position of LFA from the polyacrylamide gel was markedlydependent upon the ionic strength of the eluting buffer: at 0.14 I, iteluted at a position equivalent to a protein of 15,000 MW, and at 1.04I, it eluted at a position equivalent to a protein of 37,000 MW. Thiswas apparently the result of ionic attractions between the basic proteinand the support medium since a similar behavior was observed forchymotrypsinogen A, the only standard protein utilized with a P^(I)similar to that of LFA.

Lines 1, 2 and 3 of Table V, summarize the results of the sedimentationequilibrium measurements on native LFA. Linear plots of 1n (Fringedisplacement) versus (radius)² were obtained from individual runs indilute buffer at protein concentrations between 0.5 mg/ml and 0.05mg/ml. When examine collectively, however, the weight-average molecularweights from these runs exhibited a tendency to increase slightly withincreasing initial protein concentration. Neither the presence of a30-fold molar excess of AcNeu (line 3) nor the complete absence of AcNeu(line 2) had an effect on the sedimentation equilibrium behavior of theprotein. The native molecular weight estimated for LFA was 44,000±2,000.From this molecular weight, an s°₂₀,w of 3.4 S, and a v of 0.724 mg/ml,the estimated f/f_(min) of 1.35 suggests that LFA is a globular protein.A limited number of sedimentation equilibrium measurements at or belowpH 3 suggest that like a number of other lectins the quaternarystructure of LFA is disrupted at low pH (Table V, line 4).

The molecular weights of the constituent polypeptide chains of LFA wereestimated by sedimentation equilibrium in 6M Gdm-Cl with and withoutreduction of disulfide bonds. In both cases (Table V, lines 7 and 8),the data were consistent with two polypeptide chains of the samemolecular weight which are held together in the native molecule bynoncovalent interactions. The circular dichroic spectrum of LFA in 6MGdm-Cl (FIG. 4B, curve 3) supports the assumption that all noncovalentinteractions in the protein were broken and that the minimal subunit hadbeen obtained. These results are also supported by empirical molecularweight estimation methods in denaturing solvents (Table V, lines 9, 11).A comparison of the gel chromatographic behavior in 6M Gdm-Cl betweenreduced and unreduced LFA suggest that there is only limited restraintimposed on the polypeptide by intrachain disulfide bonds (Table VI,lines 9, 10). This observation is consistent with the half-cystinecontent of the protein (Table V) which suggests a maximum of only 1.5disulfide bonds per 100 amino acid residues.

When LFA was exposed to a 40-fold molar excess of its receptor ligand,AcNeu, the only change observed in the circular dichroic spectrum of theprotein was a diminution of about 10% in the amplitudes of theellipticites between 240 and 285 nm (cf. curves 1 and 3 of FIG. 5A).Though this is a subtle change, it is consistent with previousobservations on other lectins where the carbohydrate(s) for which thelectin is specific elicit a change in the near UV circular dichroicspectra. The far UV circular dichroic spectrum of LFA from which allfree AcNeu had been removed exhibited no measurable change upon exposureof the lectin to a 40-fold molar excess of AcNeu.

                  TABLE V                                                         ______________________________________                                        Macromolecular Properties of Slug Lectin.                                     Physical Measurement       Magnitude                                          ______________________________________                                        1.  Molecular weight by sedimentation equilibrium,                                                           44,000 ±                                        dilute buffer, pH 7.5; 4 concentrations, 3                                                               2,000 (6).sup.a                                    rotor speeds                                                              2.  Molecular weight by sedimentation equilibrium,                                                           42,500                                             AcNeu-free protein, dilute buffer 7.5                                     3.  Molecular weight by sedimentation equilibrium,                                                           43,800 (3)                                         dilute buffer plus 30 mM N--acetylneuraminic                                  acid, pH 7.5; 3 concentrations                                            4.  Molecular weight by sedimentation equilibrium,                                                           18,500.sup.b                                       dilute buffer plus 30 mM N--acetylneuraminic                                  acid, pH 2.05                                                             5.  s°.sub.20,W by sedimentation velocity, dilute                                                     3.4 S ±                                         buffer, pH 7.5             0.2 S                                          6.  f/f.sub.min                1.35                                           7.  Subunit molecular weight by sedimentation                                                                22,000-                                            equilibrium, S--S reduced in 6 M Gdm.Cl                                                                  26,000.sup.c (4)                               8.  Subunit molecular weight by sedimentation                                                                21,500-                                            equilibrium, S--S unreduced in 6 M Gdm.Cl                                                                23,000.sup.c                                   9.  Re by gel chromatography in 6 M Gdm.Cl,                                                                  41.4 Å                                         S--S reduced               Mr = 19,100                                    10. Re by gel chromatography in 6 M Gdm.Cl,                                                                  40.8 Å                                         S--S unreduced                                                            11. Subunit molecular weight by NaDodSO.sub.4 gel                                                            22,500                                             electrophoresis                                                                ##STR1##                   2.1 ± 0.2cm.sup.2 mg.sup.-1(3).sup.a       13. -v from amino acid composition                                                                           0.724 ml/g                                     14. pI from isoelectric focusing                                                                             9-9.5                                          ______________________________________                                         .sup.a number of determinations                                               .sup.b This includes a correction for the Donnan effect; a timedependent      decrease in M.sub.app was observed. This latter observation suggests an       acidcatalyzed chain hydrolysis.                                               .sup.c This range of values includes the precision of the ultracentrifuge     data and the uncertainty of φ' in 6 M Gdm.Cl; values equal to, or 0.0     ml/g less than, -v were used for φ'.                                 

The sialoproteins, bovine submaxillary mucin and bovin fetuin, arepotent inhibitors of the slug lectin. Hemagglutination assays wereperformed as in FIG. 1 except that various amounts of the indicatedprotein in 0.5M tris-Cl-0.1M NaCl, pH 7.5 were added to the assay. Thebovine submaxillary mucin preparation used contained only 5%N-acetylneuraminic acid as determined by the thiobarbituric acid assayof Warren (J. Biol. Chem., Vol. 234, pp. 1971-1975 (1959) and thereforewas rather impure since the mucin has been reported to contain 36%N-acetylneuraminic acid (Gottschalk et al, "Glycoproteins", ElsevierPub. Co., N.Y., p. 818 (1972)). However, the mucin preparation was amore potent inhibitor of the hemagglutinating activity of the sluglectin than was highly purified fetuin. (See FIG. 3). The bovinsubmaxillary mucin gave a 50% inhibition at 10 μg/ml and the fetuin gavea 50% inhibition at approximately 55 μg/ml. The increased inhibitoryactivity of the bovine submaxillary mucin may be due to impurities inthe preparation or to an increased affinity of the slug lectin for thesialoglycopeptides found in the mucin. Asialylation of bovinesubmaxillary mucin and fetuin by incubation at 80° for one hour in 0.1NH₂ SO₄ resulted in complete loss of inhibitory activity (FIG. 3).Furthermore, double diffusion of slug lectin in an agar gel againstbovine submaxillary mucin or fetuin yielded precipitin lines which werelost when the mucin or fetuin were asialylated.

Coupling of Slug Lectin to Sepharose 4B: Five ml of affinity purifiedslug lectin at 1.0 mg/ml was dialyzed against 500 ml of 0.05M NaPO₄--0.1M NaCl, pH 6.8. The dialyzed slug lectin was coupled to 5 ml ofSepharose 4B by the cyanogen bromide activation procedure describedpreviously for the coupling of BSM to Sepharose 4B.

Affinity Purification of Sialoproteins: The slug lectin-Sepharose 4B waspoured into a one cm diameter column and washed with 0.05M Tris-Cl-0.10MNaCl, pH 7.5 to remove non-coupled slug lectin. An aliquot of serum orplasma (˜0.14 ml) containing 10 mg of protein was diluted to 2.0 milwith the above tris-saline buffer and run onto the column of sluglectin-Sepharose 4B at a flow rate of ˜0.5 ml/min. The column was washedwith tris-saline buffer until non-bound proteins were eluted. Thensialoproteins were eluted by further washing the column with tris-salinecontaining 0.01M N-acetylneuraminic acid. Approximately 2.0 ml or 6 min.fractions were collected. Fractions containing non-bound proteins andfractions containing sialoproteins were combined separately.

Separation of Sialoproteins by Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis: An aliquot of the sialoproteins and non-sialoproteins,from the above affinity purification, containing 20 μg of protein wasdiluted to 0.27 ml with the tris-saline buffer. To this was added 0.03ml of an 8-fold concentrated sample buffer (Laemmli, Nature (London),Vol. 227, pp. 680-685, (1970)) and the mixture was heated in a boilingwater bath for 10 min. and then allowed to cool to room temperature. A200 μl aliquot of the above solution was applied to a well in a 1.5 mmthick slab polyacrylamide gel and electrophoressed at 20 mA until thebromphenol blue had migrated to the bottom of the gel (˜6 hr). The gelwas removed, fixed in 40 ml of 12.5% trichloroacetic acid and 2.5 ml ofa 0.25% aqueous solution of G250 Coomassie blue was added to thetrichloroacetic acid to stain the sialoprotein bands. The stained gelwas photographed, dried and stored. This provides a means of directlycomparing the sialoproteins and/or sialopeptides present in the serum orplasma of various individuals (see FIG. 6). [Lanes 1,3, and 5, Non-boundor non-sialoproteins from serum samples. Lanes 2,4, and 6, Bound orsialoproteins from serum samples. Lanes 7 and 8, whole serum samples,i.e., non-fractionated]. The same procedure also provides a method forthe separation and comparison of the sialoproteins or sialopeptides ofother biological fluids such as urine or those solubilized from cellularmembranes.

I claim:
 1. A diagnostic method for determining the content of sialicacid or sialoprotein in a biological liquid comprising contacting thesaid biological liquid with a predetermined quantity of a substantiallyhomogeneous lectin of the slug Limax flavus having a high degree ofhemagglutinative inhibitory specificity and selectivity for sialic acidor sialoprotein having an approximate amino acid composition:

    ______________________________________                                                     Moles per 22,000 g of Lectin                                     Amino Acid   (Nearest integer)                                                ______________________________________                                        Asx          26                                                               Thr          13                                                               Ser          13                                                               Glx          14                                                               Pro          4                                                                Gly          22                                                               Ala          16                                                               Val          7                                                                Met          2                                                                Ile          7                                                                Leu          15                                                               Tyr          10                                                               Phe          10                                                               His          7                                                                Lys          17                                                               Trp          6                                                                Arg          8                                                                Cys          6                                                                ______________________________________                                    

and measuring the degree of hemagglutination effected by said lectin andcomparing said measurement with the degree of hemagglutination effectedwith contacting an identical quantity of said lectin with a similarbiological fluid containing a known amount of sialic acid orsialoprotein.
 2. The diagnostic method of claim 1 wherein the degree ofhemagglutination is measured by determining the optical density of thetreated biological liquid.
 3. The diagnostic method of claim 1 whereinsaid biological liquid is whole blood.
 4. The diagnostic method of claim1 wherein said biological liquid is plasma.
 5. The diagnostic method ofclaim 1 wherein said biological liquid is serum or urine.
 6. Thediagnostic method of claim 1 wherein said liquid is tissue or cellularhomogenate.